| Summary: | Pseudomonas aeruginosa is an opportunistic human pathogen that produces an extensive array of virulence determinants, many of which are regulated via quorum-sensing (QS) in a cell-density dependent manner and play an important role in the outcome of infection. QS controls virulence factor production via two signalling systems driven by either N-acylhomoserine lactone (AHL) or 2-alkyl-4-quinolone (AQ) signal molecules. The blocking of QS has been proposed as a potential future antibacterial drug strategy, therefore this study aimed to further the knowledge of QS in P. aeruginosa, investigating how QS is connected to other regulatory networks and virulence, and the potential of alternative methods of blocking QS. The use of probiotic bacteria to inhibit growth, QS and virulence factor production in P. aeruginosa was investigated. Nine probiotic species of bacteria were unable to inhibit QS, degrade QS signal molecules, or affect the production of virulence determinants, biofilm formation or attachment. Interestingly, the effects observed were mainly attributed to the MRS growth medium in which probiotics were grown as this medium showed significant antibacterial and quorum quenching properties. The function and the regulation of two genes, PA2383 and PA2384, which have been suggested to have roles in QS and iron-regulation, were investigated. PA2383 and PA2384, predicted to be in the same operon, were regulated at the transcriptional level both by a mechanism dependent upon iron availability and by AQ-like molecules with either QS or iron chelation abilities. Furthermore, these genes were involved in an autoregulatory feedback loop in which PA2384 either repressed or activated the transcription of the operon under iron-replete or iron-limited conditions, respectively. Additionally, positive post-transcriptional regulation was exerted on PA2384 by RsmA, itself under the control of GacS-GacA. PA2383 and PA2384 were found to regulate the expression of QS-dependent genes (including pqsH, pqsL, rhll, rhiR, last, phzA1) and genes required for iron uptake (pvds, pvdE, pirA and pfeA). PA2383 and PA2384 may therefore provide a link between QS, iron homeostasis and the GacS-GacA global regulatory system. Forty-nine P. aeruginosa clinical wound isolates from colonised patients were characterised to investigate variation and search for correlations in QS, virulence and antibiotic resistance. The isolates were found to produce varying levels of AHLs and AQs, and interestingly AHL proficient but AQ deficient isolates were also identified. Pyocyanin, LasB elastase and universal protease production in the isolates also varied. Some positive correlation between 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and pyocyanin levels, and N-(3-oxo-dodeconyl)-homoserine lactone (3-oxo-C12-HSL) and LasB elastase/protease levels was also seen, however no correlation between QS signal production and swarming or attachment capabilities was found. High antibiotic susceptibility was seen in the isolates to aminoglycosides, but also high resistance to meropenem. The antibiotic susceptibility profile did not show great variability across the isolates and as such, no correlation between QS and antibiotic resistance was evident. In summary, studies have shown that the QS systems in P. aeruginosa may be a good target for novel antibacterial treatments. A proposed source for quorum quenching activity, probiotic bacteria, was investigated however was found to be ineffective against P. aeruginosa. This work further aimed to advance the knowledge of the QS systems to both discover how they are linked to other complex regulatory systems in P. aeruginosa, and to determine the characteristics and QS abilities of P. aeruginosa wound isolates for which quorum quenching treatments may one day act upon. This work gives new insight into the QS regulatory networks that will advance the knowledge needed for the development of quorum quenching agents.
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